20. The evaluation of the cell growth is important to determine the
lag phase, exponential phase, and stationary phase of the cell
suspension culture. This is essential in order to identify the best
time interval in which to perform experiments (usually with
mid-exponential phase cells) and the subculturing time, when
the cell culture must be renewed in fresh culture medium (i.e.,
with late-exponential phase cells).
21. If multiple measurements have to be carried out, weigh separately each tube, because there can be small differences in the
weight between tubes.
22. Boxes of sterile tips with a wide bore can be prepared in
advance, by cutting the tip ends with a sterile scalpel and then
autoclaving them. Before the transfer, clean the micropipette
with a paper towel and denatured alcohol. Use extreme caution
while you enter the flask with the micropipette: avoid touching
the inner glass sides, in order to minimize contamination risks.
23. Heterotrophic calli can be maintained in vitro almost indefinitely, by subculturing them once a month onto new plates.
This is particularly useful, because they represent a reservoir of
cells ready to use when cell suspension cultures need to be set
up again, for example, in the unfortunate event of a fungal/
bacterial contamination.
24. Changes in sucrose concentration must be stepwise and the
subcultures have to be kept in the same concentration of
sucrose for 2–3 weeks.
25. In our hands, total deprivation of sugar in the culture medium
is not advisable because, although not compromising the photosynthetic activity of the cell culture, it causes a drastic reduction of the cell growth rate, making the performance of weekly
experiments unfeasible.
26. Determine the PCV as detailed in Note 18. Then transfer
the cell culture volume corresponding to 2.5 mL PCV in the
50 mL tube, centrifuge at 500 g for 2 min, and discard the
supernatant.
27. Prepare this filter system by using non-sterile labware, wrap it
in aluminum foil, and sterilize by autoclaving.
28. If required, a counting assay can be carried out to determine
protoplasts concentration, viability, and yield by using a light
microscope, Trypan blue staining, and a Bu ¨rker chamber, as
previously described [3].
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Enrico Cortese et al.
lag phase, exponential phase, and stationary phase of the cell
suspension culture. This is essential in order to identify the best
time interval in which to perform experiments (usually with
mid-exponential phase cells) and the subculturing time, when
the cell culture must be renewed in fresh culture medium (i.e.,
with late-exponential phase cells).
21. If multiple measurements have to be carried out, weigh separately each tube, because there can be small differences in the
weight between tubes.
22. Boxes of sterile tips with a wide bore can be prepared in
advance, by cutting the tip ends with a sterile scalpel and then
autoclaving them. Before the transfer, clean the micropipette
with a paper towel and denatured alcohol. Use extreme caution
while you enter the flask with the micropipette: avoid touching
the inner glass sides, in order to minimize contamination risks.
23. Heterotrophic calli can be maintained in vitro almost indefinitely, by subculturing them once a month onto new plates.
This is particularly useful, because they represent a reservoir of
cells ready to use when cell suspension cultures need to be set
up again, for example, in the unfortunate event of a fungal/
bacterial contamination.
24. Changes in sucrose concentration must be stepwise and the
subcultures have to be kept in the same concentration of
sucrose for 2–3 weeks.
25. In our hands, total deprivation of sugar in the culture medium
is not advisable because, although not compromising the photosynthetic activity of the cell culture, it causes a drastic reduction of the cell growth rate, making the performance of weekly
experiments unfeasible.
26. Determine the PCV as detailed in Note 18. Then transfer
the cell culture volume corresponding to 2.5 mL PCV in the
50 mL tube, centrifuge at 500 g for 2 min, and discard the
supernatant.
27. Prepare this filter system by using non-sterile labware, wrap it
in aluminum foil, and sterilize by autoclaving.
28. If required, a counting assay can be carried out to determine
protoplasts concentration, viability, and yield by using a light
microscope, Trypan blue staining, and a Bu ¨rker chamber, as
previously described [3].
184
Enrico Cortese et al.